Angiogenesis is the process of generating new capillary blood vessels and involves an interplay between cells and soluble factors. Thus, activated endothelial cells migrate and proliferate to form new vessels, which are surrounded by layers of periendothelial cells; small blood vessels are surrounded by pericytes and large blood vessels are surrounded by smooth muscle cells.
Numerous factors are known to regulate angiogenesis. These include soluble factors and tissue oxygen. Factors known to positively regulate angiogenesis include Vascular Endothelium Growth Factor (VEGF), basic Fibroblast Growth Factor (bFGF), acidic FGF/FGF-1 and hypoxia-inducible factor-1α (HIF-1α). Hypoxia induces the expression of several gene products such as erytropoietin, VEGF, bFGF and glycolytic enzymes.
Angiogenesis-dependent pathologies result from disregulated angiogenesis, i.e., excessive amounts of new blood vessels or insufficient number of blood vessels. Insufficient angiogenesis is related to a large number of diseases and conditions, such as coronary artery diseases, delayed wound healing, delayed ulcer healing, reproduction associated disorders, arteriosclerosis, myocardial ischemia, peripheral ischemia, cerebral ischemia, retinopathy, remodeling disorder, von Hippel-Lindau syndrome, diabetes and hereditary hemorrhagic telengiectasia. On the other hand, excess of angiogenesis is characteristic to cancerous cells and cancer metastasis.
Common treatment of ischemic diseases (e.g., peripheral artery diseases such as critical limb ischemia, coronary artery disease) involves mechanical revascularization by percutaneous techniques or a bypass surgery using arterial and venous conduits as grafts onto the coronary arterial tree. However, these treatment modalities have significant limitations in individuals with diffuse atherosclerotic disease or severe small vessel coronary artery disease, in diabetic patients, as well as in individuals who have already undergone surgical or percutaneous procedures. For these reasons, therapeutic angiogenesis, aimed at stimulating new blood vessel growth, is highly desirable.
The therapeutic concept of angiogenesis therapy is based on the premise that the existing potential for vascular growth inherent to vascular tissue can be utilized to induce the development of new blood vessels under the influence of the appropriate angiogenic molecules.
Animal studies have proven the feasibility of enhancing collateral perfusion and function in experimental models of acute and chronic ischemia via exogenous angiogenic compounds (Sun Q, Chen R R, Shen Y, Mooney D J, Rajagopalan S, Grossman P M. Sustained vascular endothelial growth factor delivery enhances angiogenesis and perfusion in ischemic hind limb. Pharm. Res. 2005; 22, 1110-6). In addition, synthetic peptides encompassing portions of the human FGF and VEGF proteins were described to efficiently agonize or antagonize the biological functions of the growth factor family members. Furthermore, screening a combinatorial phage display library of random 12-mer peptides resulted in isolation of specific peptides capable of binding the cell-surface of endothelial cells and triggering angiogenic processes which included endothelial cell-proliferation and vascularization (PCT Pub. WO2005/039616 to the present inventors).
Members of the ADAM (A Disintegrin And Metalloproteinase) family of proteolytic enzymes are implicated in the processing of many single transmembrane-bound proteins ranging from cell surface receptors to growth factors and cytokines. The disintegrin domains in the ADAM proteins compete with extracellular proteins (ECM) on integrin binding. As such, the ADAM proteins are thought to be involved in the regulation of cell/ECM- and cell/cell-interactions in many physiological and pathophysiological conditions. In addition, the conserved metalloprotease domain in ADAM proteins is thought to be involved in shedding of biologically important cell surface proteins. Thus, it was suggested that ADAM proteases could facilitate cell migration by shedding ectodomains and by remodeling of the ECM (Trochon V., et al., 1998).
ADAM15 (also known as metargidin) is a membrane-anchored glycoprotein implicated in cell-cell or cell-matrix interactions and in the proteolysis of molecules on the cell surface or extracellular matrix. The expression level of ADAM15 was found to be elevated in numerous tissues and conditions characterized by extensive remodeling such as vascular cells, endocardium, atherosclerotic lesions, rheumatoid tissue, chondrosarcoma and atrial fibrillation and dilatation. In addition, ADAM15 is expressed in human aortic smooth muscle and cultured Umbilical Vein Endothelial Cells (HUVECs) and the ADAM15 gene was localized to human chromosome band 1q21.3 that is amplified in several types of cancers.
The possible role of ADAM15 in neovascularization was studied in mice lacking the ADAM15 gene (i.e., ADAM15 knock out mice). The ADAM15 knock out mice exhibit a major reduction in neovascularization compared to wild-type controls (Bohm B B, Aigner T, Roy B Brodie T A, Blobel C P Burkhardt H; Arthritis Rheum. 2005 52, 4 1100-9); a strongly reduced angiogenic response in a model of hypoxia-induced proliferative retinopathy; and significantly smaller tumors which develop after implantation of melanoma cells. Specific candidate substrates for ADAM15 in the context of neovascularization include Notch1 and -4, PECAM-1, VE-cadherin, TIE-2, membrane type 1 MMP and possibly also Kit-ligand. On the other hand, although ADAM15 demonstrates strong and specific interactions with hematopoietic Src family kinases, which are known to be required for VEGF-mediated angiogenesis, nor VEGF or bFGF induce changes in ADAM15 expression in HUVECs.
Inhibition of ADAM15 by specific antibodies or the metalloprotease inhibitor BB3103 resulted in blockage of human mesangial cell migration and suggested that the metalloprotease activity is essential for this process.
The glucose-regulated protein (GRP78) (also known as HSPA5 or BiP), is a member of the heat-shock protein-70 (HSP70) family, highly conserved molecules that act as molecular chaperones and is involved in the folding and assembly of proteins in the endoplasmic reticulum (ER). GRP78 was found to be upregulated in drug-resistant lung cancer cell lines and its expression level was inversely correlated to the microvessel density (MVD) [Koomaqi R., et al., 1999; Anticancer Res. 19(5B): 4333-6]. In contrast, inhibition of GRP78 using small interfering RNA resulted in sensitization of human breast cancer cells to etoposide-mediated cell death [Dong D., et al., 2005, Cancer Res. 65(13): 5785-91]. On the other hand, no correlation was found between the expression level of ER-stress response protein GRP78 and the resistance to hypoxia or ER stresses [Koshikawa N., et al., 2006, Oncogene 25(6):917-28]. Recently, GRP78 was found to be exposed on the cell surface of proliferating endothelial cells and stressed tumor cells and to play a key role in the anti-angiogenic and antitumor activity of Kringle 5 (K5) [Davidson D J., et al., 2005, Cancer Res. 65(11): 4663-72].